Ubiquitin Accumulation on Disease Associated Protein Aggregates Is Correlated with Nuclear Ubiquitin Depletion, Histone De-Ubiquitination and Impaired DNA Damage Response
Fig 4
Cells with Poly Q aggregates have an impaired DNA damage response, which can be partially rescued by ubiquitin over-expression.
U2OS cells were transiently transfected with Htt-Q91-Cherry, treated with DNA damaging agent neocarzinostatin and fixed at indicated time points. Cells were stained with an antibody against the early marker for DDR phosphor-gamma-H2AX. The red line indicates cells that lack aggregates and the blue line cells with aggregates. The presented experiment is a representative of three repeats (A, B). U2OS cells were co-transfected with Ubi[9]-mRFP[1] (red) and Htt-Q91-Venus treated with neocarzinostatin and fixed after one hour (C). Cells transiently transfected with Htt-Q91-Cherry, and treated with neocarzinostatin were fixed at indicated time points and stained with antibodies against the late DDR marker 53BP1 (D, E). Cells were either imaged by microscope (A, C, D, E) or analyzed by flow cytometry. The red bar indicates cells that lack aggregates and the blue bar cells with aggregates. Number of foci of 53BP1 were counted manually, error bars represent SE, three repeats (E).